Tissue development and regeneration is highly complex and dynamic, involved with extensive remodeling of cells and the extracellular matrix surrounding them inside the developing and/or injured tissues. Despite the rapid development of tissue engineering technologies to study regeneration, a major barrier still exists in our inabilit to monitor dynamic biological processes in a minimally invasive real-time fashion, which significantly reduces the clinical relevance of these techniques. Most available assessment methods are static, requiring sacrifice of experimental samples at fixed time points. Therefore, there is an unmet need for new technologies that will provide non-destructive and dynamic monitoring of the development and regeneration processes. Optical imaging in biology can be broadly classified as either ballistic imaging or diffusive imaging. The combination of fluorescent and bioluminescent probes with optoelectronics and computing techniques has led to the development of optical molecular imaging tools that allow the visualization of biologic interactions in complex, living systems over time. However, despite the great potential of optical molecular imaging, it has not yet been harnessed as an enabling technology for tissue regeneration research, due to tissue turbidity, resulting in strong scatter and absorption of light and limited penetration depth, requiring direct view of the tissue. We have recently published several seminal manuscripts describing the development of an indirect, non-destructive, cellular-level imaging instrument through a combination of fiber optic technology and an image reconstruction approach and generation of bioengineered mature and vascularized skeletal muscle tissue using combinations of fluorescently labeled cells. These achievements serve as the motivation for the current proposal, which aims to utilize the model of bioengineered skeletal muscle to develop and validate a novel optical molecular tomography platform, which could be broadly used for tissue regeneration research. We hypothesize that 1) optical imaging, photon transport modeling, and image reconstruction will allow for the non- invasive (indirect), dynamic analysis of bioengineered muscle tissue constructs~ and 2) tomography of distinct fluorescent probes will improve the examination of developing bioengineered muscle constructs, comprised of multiple cell types. We will test these hypotheses by developing a multiwell tissue culture dish equipped with fiber-based imaging system. We will first test the capacity of the imaging system to generate optical phantoms of fluorescently labeled cells and subsequently use the imaging system to assess the organization and differentiation of muscle progenitor and endothelial cells into a multicellular skeletal muscle tissue in vitro. These studies have the potential to drive a paradigm shift from static assays of cellular function in 2D culture models towards systematic analyses of 3D tissues. Achieving the goals set forth in this proposal will establish a novel technology to construct and image 3D composite bioengineered tissues and improve our understanding of tissue development and regeneration mechanisms.